Method for treating atherosclerosis or restenosis using microtubule stabilizing agent

ABSTRACT

The present invention is a method of preventing or reducing atherosclerosis or restenosis, and a pharmaceutical preparation used therefore. In particular, it is a method of preventing or reducing atherosclerosis or restenosis after arterial injury by treatment with a low dose of a microtubule stabilizing agent such as taxol or a water soluble taxol derivative. The low dose used in the present invention prevents artery blockage while minimizing any negative side effects associated with the drug.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. patent application Ser. No. 12/072,067filed Feb. 21, 2008, which is a continuation of U.S. patent applicationSer. No. 11/644,411 filed Dec. 21, 2006 (now abandoned), which is acontinuation of U.S. application Ser. No. 11/304,362 filed Dec. 14, 2005(now abandoned), which is a continuation of U.S. application Ser. No.10/272,496 filed Oct. 15, 2002 (now abandoned), which is a continuationof U.S. application Ser. No. 10/121,500 filed Apr. 11, 2002 (issued asU.S. Pat. No. 6,500,859), which is a continuation of U.S. applicationSer. No. 08/821,906 filed Mar. 21, 1997 (issued as U.S. Pat. No.6,429,232), which is a continuation of U.S. application Ser. No.08/633,185 filed Apr. 18, 1996 (issued as U.S. Pat. No. 5,616,608),which is a continuation of U.S. application Ser. No. 08/099,067 filedJul. 29, 1993 (now abandoned). The entire disclosures of all of theprior applications are considered to be part of the disclosure of thepresent application and are hereby incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to a method of treating patients at riskof developing atherosclerosis or restenosis.

More particularly, the invention relates to treatment of these patientswith a low dose taxol solution to prevent or reduce the development ofatherosclerosis or restenosis.

BACKGROUND OF THE INVENTION

Vascular disease is the leading cause of death and disability in thedeveloped world, particularly afflicting the elderly. In the UnitedStates alone, despite recent encouraging declines, cardiovasculardisease is still responsible for almost one million fatalities each yearand more than one half of all deaths; almost 5 million persons afflictedwith cardiovascular disease are hospitalized each year. The cost of thisdisease in terms of human suffering and of material resources is almostincalculable.

Atherosclerosis is the most common form of vascular disease and leads toinsufficient blood supply to critical body organs, resulting in heartattack, stroke, and kidney failure. Additionally, atherosclerosis causesmajor complications in those suffering from hypertension and diabetes,as well as tobacco smokers. Atherosclerosis is a form of chronicvascular injury in which some of the normal vascular smooth muscle cells(“VSMC”) in the artery wall, which ordinarily control vascular toneregulating blood flow, change their nature and develop “cancer-like”behavior. These VSMC become abnormally proliferative, secretingsubstances (growth factors, tissue-degradation enzymes and otherproteins) which enable them to invade and spread into the inner vessellining, blocking blood flow and making that vessel abnormallysusceptible to being completely blocked by local blood clotting,resulting in the death of the tissue served by that artery.

Restenosis, the recurrence of stenosis or artery stricture aftercorrective surgery, is an accelerated form of atherosclerosis. Recentevidence has supported a unifying hypothesis of vascular injury in whichcoronary artery restenosis along with coronary vein graft and cardiacallograft atherosclerosis can be considered to represent a muchaccelerated form of the same pathogenic process that results inspontaneous atherosclerosis (Ip, J. H., et al., (1990) J Am CollCardiol, 15:1667-1687; Muller, D. W. M., et al., (1992) J Am CollCardiol, 19:418-432). Restenosis is due to a complex series offibroproliferative responses to vascular injury involving potentgrowth-regulatory molecules, including platelet-derived growth factor(PDGF) and basic fibroblast growth factor (bFGF), also common to thelater stages in atherosclerotic lesions, resulting in vascular smoothmuscle cell proliferation, migration and neointimal accumulation.

Restenosis occurs after coronary artery bypass surgery (CAB),endarterectomy, and heart transplantation, and particularly after heartballoon angioplasty, atherectomy, laser ablation or endovascularstenting (in each of which one-third of patients redevelopartery-blockage (restenosis) by 6 months), and is responsible forrecurrence of symptoms (or death), often requiring repeatrevascularization surgery. Despite over a decade of research andsignificant improvements in the primary success rate of the variousmedical and surgical treatments of atherosclerotic disease, includingangioplasty, bypass grafting and endarterectomy, secondary failure dueto late restenosis continues to occur in 30-50% of patients (Ross, R.(1993) Nature, 362:801-809).

As a result, a need exists for a successful chemotherapeutic therapy toreduce or prevent artery-blockage. The most effective way to preventthis disease is at the cellular level, as opposed to repeatedrevascularization surgery which can carry a significant risk ofcomplications or death, consumes time and money, and is inconvenient tothe patient.

Microtubules, cellular organelles present in all eukaryotic cells, arerequired for healthy, normal cellular activities. They are an essentialcomponent of the mitotic spindle needed for cell division, and arerequired for maintaining cell shape and other cellular activities suchas motility, anchorage, transport between cellular organelles,extracellular secretary processes (Dustin, P. (1980) Sci. Am., 243:66-76), as well as modulating the interactions of growth factors withcell surface receptors, and intracellular signal transduction.Furthermore, microtubules play a critical regulatory role in cellreplication as both the c-mos oncogene and CDC-2-kinase, which regulateentry into mitosis, bind to and phosphorylate tubulin (Verde, F. et al.(1990) Nature, 343:233-238), and both the product of the tumorsuppressor gene, p53, and the T-antigen of SV-40 bind tubulin in aternary complex (Maxwell, S. A. et al. (1991) Cell Growth Differen.,2:115-127). Microtubules are not static, but are in dynamic equilibriumwith their soluble protein subunits, the α- and β-tubulin heterodimers.Assembly under physiologic conditions requires guanosine triphosphate(GTP) and certain microtubule associated and organizing proteins ascofactors; on the other hand, high calcium and cold temperature causedepolymerization.

Interference with this normal equilibrium between the microtubule andits subunits would therefore be expected to disrupt cell division andmotility, as well as other activities dependent on microtubules. Thisstrategy has been used with significant success in the treatment ofcertain malignancies. Indeed, antimicrotubule agents such as colchicineand the vinca alkaloids are among the most important anticancer drugs.These antimicrotubule agents, which promote microtubule disassembly,play principal roles in the chemotherapy of most curable neoplasms,including acute lymphocytic leukemia, Hodgkin's and non-Hodgkin'sLymphomas, and germ cell tumors, as well as in the palliative treatmentof many other cancers.

The newest and most promising antimicrotubule agent under research istaxol. Taxol is an antimicrotubule agent isolated from the stem bark ofTaxus brevifolia, the western (Pacific) yew tree. Unlike otherantimicrotubules such as colchicine and the vinca alkaloids whichpromote microtubule disassembly, taxol acts by promoting the formationof unusually stable microtubules, inhibiting the normal dynamicreorganization of the microtubule network required for mitosis and cellproliferation (Schiff, P. B., et al. (1979) Nature 277:665; Schiff, P.B., et al. (1981) Biochemistry 20:3247). In the presence of taxol, theconcentration of tubulin required for polymerization is significantlylowered; microtubule assembly occurs without GTP and at lowtemperatures, and the microtubules formed are more stable todepolymerization by dilution, calcium, cold, and inhibitory drugs. Taxolwill reversibly bind to polymerized tubulin, and other tubulin-bindingdrugs will still bind to tubulin even in the presence of taxol.

Taxol has one of the broadest spectrum of an antineoplastic activity,renewing serious interest in chemotherapeutic strategies directedagainst microtubules (Rowinsky, E. K., et al. (1990) Jrnl. of the Nat'l.Cancer Inst., 82:1247-1259). In recent studies, taxol has shownsignificant activity in advanced and refractory ovarian cancer (Einzig,A. I., et al. (1992) J. Clin. Oncol., 10:1748), malignant melanoma(Einzig, A. I. (1991) Invest. New Drugs, 9:59-64), as well as in cancersof the breast (Holmes, F. A., et al. (1991) JNCI, 83:1797-1805), headand neck, and lung.

Taxol has been studied for its effect in combating tumor growth inseveral clinical trials using a variety of administration schedules.Severe allergic reactions have been observed following administration oftaxol. However, it is has been demonstrated that the incidence andseverity of allergic reactions is affected by the dosage and rate oftaxol infusion (Weiss, R. B., et al. (1990) J. Clin. Oncol. 8:1263).

Cardiac arrhythmias are associated with taxol administration, and likeallergic reactions, their incidence is affected by the dosage and rateof taxol administration. Sinus bradycardia and Mobitz II arrhythmia willdevelop in approximately 40% and 5% of patients, respectively, beginning4-6 hours after the start of a taxol infusion, and continuing for 4-8hours after its completion. In most patients, the abnormal rhythm istransient, asymptomatic, hemodynamically stable, and does not requirecardiac medications or electrical pacing. Additionally, it has beenobserved that the incidence of severe cardiac events is low in patientsreceiving taxol alone. Thus, infusion times up to 24 hours have beenused in treatment with taxol to decrease the incidence of toxicity andallergic reaction to the drug.

During angioplasty, intraarterial balloon catheter inflation results indeendothelialization, disruption of the internal elastic lamina, andinjury to medial smooth muscle cells. While restenosis likely resultsfrom the interdependent actions of the ensuing inflammation, thrombosis,and smooth muscle cell accumulation (Ferrell, M., et al. (1992) Circ.,85:1630-1631), the final common pathway evolves as a result of medialVSMC dedifferentiation from a contractile to a secretory phenotype. Thisinvolves, principally, VSMC secretion of matrix metalloproteinasesdegrading the surrounding basement membrane, proliferation andchemotactic migration into the intima, and secretion of a largeextracellular matrix, forming the neointimal fibropoliferative lesion.Much of the VSMC phenotypic dedifferentiation after arterial injurymimics that of neoplastic cells (i.e., abnormal proliferation,growth-regulatory molecule and protease secretion, migration andbasement invasion).

Although others have investigated the use of the antimicrotubule agentcolchicine in preventing restenosis, opposite conclusions have beenreported (See Currier, et al., “Colchicine Inhibits Restenosis AfterIliac Angioplasty In The Atherosclerotic Rabbit” (1989) Circ., 80:11-66;O'Keefe, et al., “Ineffectiveness Of Colchicine For The Prevention OfRestenosis After Coronary Angioplasty” (1992) J. Am. Coll. Cardiol.,19:1597-1600). The art fails to suggest the use of a microtubulestabilizing agent such as taxol in preventing or reducing this disease.Thus, the method of the present invention is to prevent or reduce thedevelopment of atherosclerosis or restenosis using a microtubulestabilizing agent such as taxol or a water soluble taxol derivative.This microtubule stabilizing mechanism of atherosclerosis or restenosisprevention is supported by the analogous results in experiments oncellular proliferation and migration using taxol and ³H₂O (deuteriumoxide), which exert comparable microtubule effects via differentunderlying mechanisms.

Accordingly, an object of this invention is to provide a method toreduce or prevent the development of atherosclerosis or restenosis usingtreatment with a drug which promotes highly stabilized tubule formation.

An additional object of this invention is to provide a method ofpreventing or reducing atherosclerosis or restenosis using apharmaceutical preparation containing a low dosage of taxol or watersoluble taxol derivative.

All references cited are herein incorporated by reference.

SUMMARY OF THE INVENTION

In accordance with these and other objects of the present invention, amethod of preventing or reducing atherosclerosis or restenosis isprovided, which comprises treatment with a therapeutically effectiveamount of a microtubule stabilizing agent such as taxol or a watersoluble taxol derivative. A therapeutically effective amount of agent isan amount sufficient to prevent or reduce the development ofatherosclerosis or restenosis.

This method provides an effective way of preventing or reducing thedevelopment of atherosclerosis or restenosis in those patientssusceptible to such disease. Additionally, because of the low dose ofchemotherapeutic agent used, the chance of a patient developing adversereactions is potentially reduced.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 depicts the taxol induced impairment of the ability of VSMC toinvade filters coated with basement membrane proteins, and taxolinhibition of cultured VSMC [³H]-thymidine incorporation.

FIG. 2 shows taxol inhibition of smooth muscle cell neointimalaccumulation after balloon catheter injury of the rat carotid artery.

FIG. 3 depicts deuterium oxide dose-dependent inhibition of VSMCchemoinvasion, and deuterium oxide inhibition of cultured VSMCbromodeoxyuridine (BrDU) incorporation.

FIG. 4 shows concentrations of taxol caused dose-dependent microtubulebundling in VSMC's cultured on plastic.

FIG. 5 shows deuterium oxide induced microtubule bundling in culturedVSCM's.

DETAILED DESCRIPTION OF THE INVENTION

The practice of an embodiment in the present invention may beaccomplished via several alternative drug delivery routes, such asintraperitoneal or subcutaneous injection, continuous intravenousinfusion, oral ingestion or local (direct) delivery, or a combination oftwo or more. When formulating a solution for injection or continuousinfusion, one must first prepare a taxol solution. Taxol is suppliedthrough CTEP, DCT, NCI (IND #22850) as a concentrated solution, 6 mg/ml,in 5 ml vials (30 mg/vial) in a polyoxyethylated castor oil (CremophorEL®) 50% and dehydrated alcohol, USP (50%) vehicle. The intact vialsshould be stored under refrigeration and diluted prior to use. Whendiluted in either 5% Dextrose Injection or 0.9% Sodium Chloride, taxolconcentrations of 0.3-1.2 mg/ml are physically and chemically stable forat least 12 hours at room temperature. (NCI Investigation Drugs;Pharmaceutical Data (1990)). It has also been demonstrated that taxolconcentrations of 0.6 mg/ml diluted in either D5W or NS and 1.2 mg/mldiluted in NS prepared in polyolefin containers are stable for at least25 hours at ambient temperatures (20-23° C.). (Waugh, et al. (1990) Am.J. Hosp. Pharm. 48, 1520). Although these concentrations have exhibitedstability for the above periods of time, they are not meant to limit thepractice of the present invention wherein any concentration of taxol maybe utilized.

All solutions of taxol exhibit a slight haziness directly proportionalto the concentrations of drug and time elapsed after preparation.Formulation of a small number of fibers in the solution (withinacceptable limits established by the USP Particulate Matter Test forLVP's) has been observed after preparation of taxol infusion solutions.While particulate formation does not indicate loss of drug potency,solutions exhibiting excessive particulate matter formation should notbe used. Therefore, when administering via continuous infusion, in-linefiltration may be necessary and can be accomplished by incorporating ahydrophilic, microporous filter with a pore size no greater than 0.22microns (IVEX-HP In Line Filter Set-SL, 15″, Abbott model #4525 orequivalent) into the fluid pathway distal to an infusion pump.

Taxol must be prepared in non-plasticized solution containers (e.g.,glass, polyolefin, or polypropylene) due to leaching ofdiethylhexylphthlalate (DEHP) plasticizer from polyvinyl chloride (PVC)bags and intravenous tubing. Taxol must not be administered through PVCintravenous or injection sets. Therefore, polyolefin- orpolyethylene-line sets, such as IV nitroglycerin sets (or equivalent)should be used to connect the bottle or bag (containing the taxolsolution for a continuous infusion) to the IV pump, a 0.22 micron filteris then attached to the IV set, and then may be directly attached to thepatient's central access device. If necessary, a polyolefin-lineextension set (Polyfin™ Extension Set, MiniMed Technologies, Model #126)can be used to provide additional distance between the IV pump and thepatient's central access device.

One category of taxol use would encompass the prevention of recurrentstenosis (restenosis) post therapeutic coronary- or peripheral-arteryangioplasty or atherectomy, after coronary bypass graft or stentsurgery, or after peripheral vascular surgery (e.g., carotid or otherperipheral vessel endarterectomy, vascular bypass, stent or prostheticgraft procedure). A human dosing schedule can consist of (but not belimited to) 24-hour continuous IV pretreatment with up to about 0.5-2mg/kg (20-80 mg/m²) prior to the vascular procedure, about 0.25-2 mg/kg(10-80 mg/m²) continuous IV infusion over the 24 hours post-procedure,then about 0.25-2 mg/kg (10-80 mg/m²) continuous IV infusion over 24hours every 21 days for 1 to 6 cycles. Such a dosage is significantlylower than that used to treat human cancers (approximately 4-6 mg/kg).

Another category of taxol use would encompass the primary prevention, orthe attenuation, of vascular disease (atherosclerosis) development.Certain of these applications (examples of which include the preventionof cardiac allograft (transplant) atherosclerosis, the multi-organsystem failure resulting from the vascular complications of diabetesmellitus or accelerated, medically-refractory atherosclerosis inpatients who are poor surgical candidates) may require the latertreatment cycles to be continuous low-dose (1-5 mg/m²/day) IV infusionsover 5-7 days. Each of the taxol treatments will generally requireretreatment with dexamethasone 20 mg orally 14 and 7 hours prior totaxol, diphenhydramine 50 mg IV and cimetidine 300 mg IV 30 min prior totaxol, to minimize potential episodes of allergic reaction. Additionalapplications that may not be associated with a surgical procedureinclude treatment of vascular fibromuscular dysplasia, polyarteritisnodosa, and Takayasu's arteritis. Each of the aforementionedapplications may also be amenable to selective, localized application ofsustained-release preparations of taxol (or othermicrotubule-stabilizing agent) which would enable high dosage local drugdelivery with little systemic toxicity.

Additionally, water soluble derivatives of taxol can also be used in thepresent invention. The water soluble derivatives of taxol, as describedin U.S. Pat. No. 5,157,049 to Haugwitz, et al. (incorporated herein byreference) include, but are not limited to, 2′-succinyl-taxol;2′-succinyl-taxol triethanolamine; 2′-glutaryl-taxol; 2′-glutaryl-taxoltriethanolamine salt; 2′-O-ester with N-(dimethylaminoethyl) glutamide;2′-O-ester with N-(dimethylaminoethyl) glutamide hydrochloride salt.These water soluble taxol derivatives can be administered in a dosageschedule analogous to that given above for taxol with the appropriatemodifications pending clarification of the pharmacokinetics of theseagents.

A pharmaceutical composition comprising an effective amount of watersoluble derivative of taxol as an active ingredient is easily preparedby standard procedures well known in the art, with pharmaceuticallyacceptable non-toxic sterile carriers, if necessary. Such preparationscould be administered orally or in injectable form, or directly to anaffected area, to a patient at risk of developing or suffering fromatherosclerosis to prevent or reduce the development of the disease.

The following examples illustrate the effectiveness of taxol (or othermicrotubule-stabilizing agents including, but not limited to, watersoluble derivatives of taxol) in inhibiting the proliferation andmigration of vascular smooth muscle cells, and should not be used tolimit the scope of the present invention.

Example 1

The in vitro ability of cultured VSMCs, pretreated with different taxolconcentrations, to invade filters coated with reconstituted basementmembrane proteins was tested to evaluate how taxol-induced microtubulebundling would impair cell processes necessary for in vivo neointimalformation.

Vascular Smooth Muscle Cells (VSMCs) were isolated bycollagenase/elasase enzymatic digestion of the medial layers of the rataorta obtained from 6 month old Wistar rats. The cells were maintainedin culture with 10% fetal calf serum, high glucose DMEM, and amino acidsupplement. Cell cultures were maintained at 37° C. in 5% CO₂.

After 18-hour taxol pre-treatment in culture, cells were fixed in 3.7%formalin, permeabilized with 1% Triton X-100, and polymerized tubulinwas labelled with mouse anti-β-tubulin antibody (SMI 62 monoclonalantibody to polymerized β-tubulin, Paragon Biotec, Inc., Baltimore,Md.). Secondary labelling was achieved with silver-enhanced, 1 nmgold-conjugated rabbit anti-mouse antibody (Goldmark Biologicals,Phillipsburg, N.J.). Representative light photomicrographs from (A)control, (B) 0.1 nM taxol, (C) 1 nM taxol, and (D) 10 nM taxol treatedVSMCs are shown in FIG. 4.

Chemonivasion (Boyden chamber) assays were performed using modifiedBoyden chamber (Albini, et al. (1987) Cancer Res., 47:3239-3245),comprising an upper chamber separated from a lower chamber by a porousPVPF filter. PVPF filters (8 μm pore diameter, Nucleopore Filters,Pleasonton, Calif.) were coated and air dried consecutively withsolutions containing 100 μg/ml type I collagen, 5 μg/ml fibronectin, and5 μg reconstituted basement membrane (produced from theEnglebreth-Holm-Swarm tumor (Kleinman, et al. (1986) Biochemistry,25:312-318), producing a continuous 10 μg thick coating of matrixmaterial. Boyden chambers were assembled by adding 10 ng/ml PDGF BB inDMEM to the lower (chemoattractant) chamber. Cells (approximately200,000) suspended in DMEM containing 0.1% BSA were then added to theupper chamber. Some of the cells used in this assays were pretreated 18hours with taxol (concentration 30 pM to 100 nM) in culture. In thetaxol-treated groups, taxol was added to the upper and lower chambers atthe same concentration as that used for pretreatment. The chambers werethen incubated for 4 hours at 37° C. in a 5% CO₂ atmosphere. At the endof the incubation period, the cells were fixed and stained withhematoxylin and eosin. The cells on the upper surface (non-invaders)were mechanically removed and the cells on the underside of the filter(invaders) were counted under 400× magnification (four random fieldswere counted per filter and all experiments were run in triplicate, andeach triplicate assay was repeated at least three times on separateoccasions using different VSMC preparations). Chemotaxis was assayed inanalogous fashion in the Boyden chambers described above, except thatthe reconstituted basement membrane was omitted. This chemoinvasionassay is accepted by those skilled in the art as exhibiting highcorrelation between invasiveness in vitro and cellular behavior as itoccurs in vivo (Iwamoto, Y., et al. (1992) Advances In ExperimentalMedicine & Biology, 324:141-9).

Using the PDGF-BB as an attractant, taxol inhibited VSMC invasion withhalf-maximal inhibitory concentration of 0.5 nM. Taxol causedessentially complete inhibition at 100 nM, and significant inhibitionwas still resolvable at 30 pM (the lowest dose used) (FIG. 1). Achemotaxis assay (filter coated only with fibronectin and collagen I,without basement membrane proteins occluding the filter pores) withPDGF-BB as the attractant was performed in analogous fashion, yieldingthe identical outcome. These results demonstrate that taxol, at least atnanomolar drug levels, inhibits VSMC invasion primarily via inhibitionof locomotion and/or shape changes, rather than by inhibiting cellularsecretion of collagenases and metalloproteinases, which are known to benecessary for VSMC to penetrate basement membrane proteins in thisassay.

Gelatinase zymography was performed on the supernatants removed afterthe 4 hour conclusion of the Boyden assays described above.Gelatin-degrading proteinases secreted into the media by VSMCs wereanalyzed by non-reducing sodium dodecyl sulfate-polyacryramide gelelectrophoresis in 10% polyacrylamide gels containing 0.1% (w/v)gelatin. Following electrophoresis, the gelatinases were renatured byincubating the gel for 30 min. at 23° C. in 2.5% (v/v) Triton X-100followed by 18 hour incubation at 37° C. in 0.2 M NaCl, 5 mM CaCl₂ 0.02%Brij 35 (w/v), 50 mM Tris-HCl (pH 7.6). The gels were stained for 90minutes with 0.5% Coomassie Brilliant Blue G-250 and destained with 10%acetic acid, 40% methanol. Gelatinolytic activity was indicated by aclear band against the background of blue-stained gelatin.

These gelatinase zymography assays from the Boyden chamber invasionexperiments confirm that the level of VSMC collagenase secretion did notvary significantly over the taxol range 30 pM to 100 nM, compared tocontrol (FIG. 2, inset).

Example 2

To confirm the fact that microtubule stabilization andhyperpolymerization is the critical and sufficient factor involved intaxol-inhibition of VSMC invasiveness, the chemoinvasion (Boydenchamber) assay was run with deuterium oxide (²H₂O, heavy water).Deuterium oxide enhances microtubule/tubulin polymerization via amechanism distinct from that of taxol. A combination of the isotope andsolvent effects of deuterium oxide reversibly increases microtubulepolymerization both by reducing the critical concentration forpolymerization for αβ-tubulin heterodimers via enhanced tubulinhydrophobic interactions (Itoh, T. J., et al. (1984) Biochim. Biophys.Acta., 800:21-27), and by converting a population of unpolymerizabletubulin to the polymerizable form (Takahashi, T. C., et al. (1984) CellStruct. Funct., 9:45-52).

VSMC's were isolated by collagense/elastase enzymatic digestion of themedial layers of the rat aorta obtained from 6 month old Wistar rats.The cells were maintained in culture with 10% fetal calf serum, highglucose DMEM, and amino acid supplement. Cell cultures were maintainedat 37° C. in 5% CO₂.

In deuterium oxide-treated cells, ²H₂O (v/v) was substituted for water(H₂O) in the preparation of the DMEM from concentrated stock. After18-hour deuterium oxide pre-treatment in culture, cells were fixed in3.7% formalin, permeabilized with 1% Triton X-100, and polymerizedtubulin was labelled with mouse anti-β-tubulin antibody (SMI 62monoclonal antibody to polymerized β-tubulin, Paragon Biotec, Inc.,Baltimore, Md.). Secondary labelling was achieved with silver-enhanced,1 nm gold-conjugated rabbit anti-mouse antibody (Goldmark Biologicals,Phillipsburg, N.J.). Representative light photomicrographs from (A)control, and (B) 75% deuterium oxide treated VSMCs are shown in FIG. 5.

Chemoinvasion assays were performed using a modified Boyden chamber,consisting of an upper chamber separated from a lower chamber by aporous PVPF filter. PVPF filters (8 μm pore diameter, NucleoporeFilters, Pleasonton, Calif.) were coated and air dried consecutivelywith solutions containing 100 μg/ml type I collagen, 5 μg/mlfibronectin, and 5 μg reconstituted basement membrane (produced from theEnglebreth-Holm-Swarm tumor), producing a continuous 10 μm thick coatingof matrix material. Boyden chambers were assembled with PDGF-BB 10 ng/mlin DMEM in the lower (chemoattractant) chamber, then cells(approximately 200,000) suspended in DMEM containing 0.1% BSA were addedto the upper chamber. Some of the cells used in these assays werepretreated 18 hours with deuterium oxide (25%, 50%, or 75% v/vsubstitution for H₂) in culture. In the deuterium oxide-treated groups,²H₂O substituted DMEM (v/v) was added to the upper and lower chambers atthe same concentration as that used for pretreatment. The chambers werethen incubated for 4 hours at 37° C. in a humidified 5% CO₂ atmosphere.At the conclusion of the experiment, the filters were removed and thecells were fixed and stained with hematoxylin and eosin. After the cellson the upper surface of the filter (non-invaders) were mechanicallyremoved, the cells on the underside (invaders) were counted under 400×magnification (four random fields were counted per filter and allexperiments were run in triplicate).

Pretreating cultured VSMCs for 18 hours with 25%, 50% or 75% deuteriumoxide caused dose-dependent microtubule hyperpolymerization similar tothat observed with taxol. This treatment likewise inhibitedPDGF-mediated VSMC Boyden chamber invasion in a dose-dependent fashion,achieving half-maximal inhibition at 25% ²H₂O, and nearly completeinhibition at 75% ²H₂O (FIG. 3).

Example 3

In addition to cell recruitment and migration, the various growthregulatory molecules elaborated after arterial injury, such as PDGF andbFGF, are also implicated in mitogenesis and cellular proliferation. Tomeasure the effect of taxol on VSMC DNA synthesis, [³H]thymidineincorporation was measured. VSMCs were plated at 4.5×10⁴ on 24-wellplates. Following 5 hr. incubation in 10% FCS+DMEM, 0.5 mCi[³H]thymidine was added and the incubation continued for an additional16 hrs. Cells were washed twice with phosphate-buffered saline,extracted with 10% TCA for 2 hrs. on ice, then centrifuged at 2,000 gfor 10 mins. Supernatants were decanted and pellets were solubilized in0.5 ml of 1 N NaOH. After neutralizing with 0.5 ml of 1 N HCl,[³H]thymidine uptake was determined by a Beckman liquid scintillationcounter. VSMCs were treated with the various concentrations of taxol forboth the 18 hr. prior to the addition of the thymidine and duringthymidine incorporation. Each condition of these experiments wasperformed in triplicate.

Taxol inhibited cultured VSMC [³H]thymidine incorporation, an index ofcell division, in a dose-dependent fashion, with a half-maximalinhibitory concentration of 5 nM. Taxol caused essentially completeinhibition at 100 nM, and significant inhibition was resolvable at 1 nM(FIG. 1). That this inhibitory profile differs somewhat from that ofinvasion and chemotaxis, demonstrating one log-concentration-unit lowersensitivity but with steeper concentration-dependence, likely arisesbecause of the considerably different roles played by mircotubulesbetween these processes. Taxol also inhibited PDGF-BB-stimulated c-fosmRNA expression in this cultured

VSMC model, in a dose-dependent fashion, with a half-maximal inhibitoryconcentration of 1 nM, with essentially complete inhibition above 20 nM.Thus, inhibition of immediate early gene induction is another importantmechanism by which taxol blocks growth factor stimulation in VSMCs, andmay underlie, at least in part, the thymidine incorporation results.

Thus, taxol significantly inhibits cultured VSMC in vitro invasion andproliferation through interference with microtubule function, disruptinglocomotion and the ability to alter shape, as well as growth-factorstimulated early gene expression and cell proliferation, atconcentrations one hundred- to one thousand-fold lower than used totreat human cancer.

Example 4

Incorporation of the thymidine analog, bromodeoxyuridine (BrDU) wasmeasured to determine the effect of deuterium oxide on VSMC DNAsynthesis. VSMCs were plated at 4.5×10⁴ on 24-well plates. Following 20hr incubation in 10% FCS+DMEM at various ²H₂O concentrations, 10 μM BrDUwas added and the incubation continued for an additional 4 hr. Cellswere washed twice with phosphate-buffered saline (PBS) and fixed with100% methanol (−20° C.) for 10 minutes. The cells were incubated for 2hr with 1N HCl to denature the DNA, and subsequently washed 4 times inPBS. Mouse monoclonal BrDU antibody (Boehringer Mannheim) in 2% BSA-PBSwas incubated with cells for 1 hr. After PBS wash, goat anti-mouseantibody conjugated with alkaline phosphatase was added. Cell nucleicontaining BrDU substituted for thymidine stained red with alkalinephosphatase substrate, while all other nuclei stained blue. The fractionof BrDU-positive nuclei was compared between control (defined as 100%)and that of the deuterium oxide-pretreated groups.

The results indicated that deuterium oxide, similar to taxol, inhibitedcultured VSMC proliferation and DNA synthesis in a dose-dependentfashion, consistent with the critical balance of microtubule-tubulindynamics in VSMC proliferation.

While taxol and deuterium oxide potentially have multiple intracellulareffects, the coincidence of their parallel effects on microtubules(despite different mechanisms of action) and on VSMC functionality atmultiple levels, indicates that the common microtubule stabilizingmechanism of action is responsible for the observed functional changes.Thus, based on the results of experiments with both taxol and deuteriumoxide, it is evident that microtubules are involved in the control ofthe most critical and sensitive intracellular mechanisms necessary forVSMCs to undergo the multiple transformations involved in thedevelopment of atherosclerosis and restenosis after arterial injury,making microtubules particularly strategic targets to influence theoutcome.

Example 5

Under a protocol approved by the National Institute on Aging Animal Careand use Committee, 6 month Wistar rats from the GRC colony wereanesthetized with 20 mg/kg body weight pentobarbital, 2 mg/kg bodyweight ketamine, and 4 mg/kg body weight xylazine intraperitoneally. Theleft external carotid artery was cannulated with 2-French Fogartyembolectomy catheter, inflated with saline and passed three times up anddown the common carotid artery to produce a distending,deendothelializing injury. The animals were treated with 2 mg/kg bodyweight taxol solution or the control animals with vehicle alone (13.4ml/kg body weight per day of 1:2:2:165 DMSO:Cremophor EL:Dehydratedethanol:phosphate buffered saline) by intraperitoneal injectionbeginning 2 hours after injury. The taxol solution or vehicle alone wasadministered once daily, as an intraperitoneal injection, for the next 4days. After 11 days the animals (8 taxol-treated and 10 vehicle-treated)were anesthetized as above and the carotid artery was isolated and fixedin 10% buffered formalin and embedded in paraffin. Cross sections of thecarotids were mounted on microscope slides and stained with hematoxylinand eosin stain. The image of the carotid artery was projected onto adigitizing board and the cross sectional areas of the intima and themedia were measured. The results are shown in FIG. 2. As indicated inthe prior art (Ferns, G. A. A. et al. (1991) Science, 253:1129-1132) therat carotid artery injury model of restenosis can be useful, in thestudy of human restenosis, and indicate potential therapeutic action inhumans.

Quantitative analysis of injured carotid segments showed that taxoltreatment reduced the neointimal area by 70% compared to vehicle treatedanimals (Table I) (*P <0.001; †P=NS; ‡P<0.001). Several of thetaxol-treated animals showed virtually no discernable neointima (in thepresence of denuded endothelium, proving injury), while all vehicletreated animals demonstrated at least modest neointimal thickening.

While the in vivo systemic taxol dose used in these experiments (2mg/kg) is significantly lower than that ordinarily used to treat humancancers (approximately 3-6 mg/kg), dramatically lower systemic dosingwith sustained or even improved efficacy could be possible combining apretreatment regimen with the optimal treatment duration. Furthermore,since the goal of therapy is to keep the “activated” VSMCs in check, orpreferably to prevent activation in the first place, until the stimulusfor growth and migration has resolved (rather than causing cytotoxicityresulting in cell death), the goal of short-term therapy with limitedtoxicity may be possible in humans. Ultimately, local sustained-releasedelivery systems may offer the best solution to prevent restenosispost-angioplasty, enabling high local concentrations of drug deliveryand essentially eliminating problems of systemic toxicity. Drug deliverysystems that can be valuable include drug-impregnated polymer-coatedmetallic stents, biodegradable drug-eluting polymer stents, andgenetically primed endothelial cells to coat metallic stents or bedelivered directly as a local endothelial cell covering. (Muller, D. W.M. et al. (1991) JACC 17:126b-131b). These systems allow safe use of achemotherapeutic agent without systemic side effects. Alternatively,treatment may involve a period of pretreatment (i.e., before vascularsurgery) via continuous intravenous infusion for a period of time,followed by a different therapy during (local, direct delivery) or after(oral, injection) surgery.

The above examples teach taxol's (or other microtubule-stabilizing agentincluding, but not limited to, water soluble derivatives of taxol)potential beneficial uses to prevent artery blockage and thereby reducethe possibility of, or prevent, heart attacks, strokes, kidney failureand renal dialysis, blindness, limb amputations, nerve loss, need forcorrective vascular surgery/angioplasty or organ transplantation, andpremature and permanent disability requiring chronic hospitalization.The invention has been described in detail, but it will be understoodthat the invention is capable of other different embodiments. As isreadily apparent to those skilled in the art, variations andmodifications can be affected within the spirit and scope of theinvention. Accordingly, the foregoing disclosure and description are forillustrative purposes only, and do not in any way limit the invention,which is defined only by the claims.

TABLE I Group Intima (mm²) Media (mm²) I/M Vehicle 0.09 ± 0.01 0.14 ±0.01 0.66 ± 0.08 Taxol 0.03 ± 0.01  0.16 ± 0.02^(†)  0.18 ± 0.04^(‡)

1. A method of inhibiting or reducing restenosis or atherosclerosis in apatient comprising: infusing a patient with a pharmaceutical preparationcomprising a therapeutically effective amount of taxol or a taxolderivative, wherein the pharmaceutical preparation is in-line filteredduring infusion.
 2. The method of claim 1, wherein the taxol or taxolderivative is administered at a dose lower than a dose used to treathuman cancers.
 3. A method of inhibiting or reducing restenosis oratherosclerosis in a patient comprising: administering to the patientabout 0.5 to about 2 mg/kg of taxol or a taxol derivative over about a24 hour time period prior to vascular surgery; administering to thepatient about 0.25 to about 2 mg/kg of taxol or a taxol derivative overabout a 24 hour time period after the vascular surgery; and thenadministering to the patient about 0.25 to about 2 mg/kg of taxol or ataxol derivative over about a 24 hour time period every 21 days for 1 to6 cycles.
 4. The method of claim 1, wherein each administration of taxolor a taxol derivative is via continuous intravenous infusion.